Congenital heart defects (CHD) are the leading cause of mortality in live-born infants (1, 2). Hypoplastic Left Heart Syndrome (HLHS) is a rare CHD that requires several major reconstructive surgeries (3). However, the reconstructed heart does not replicate normal anatomy and function, and many patients suffer from serious secondary complications throughout life (4). Cardiac tissue engineering is especially promising for treating HLHS by creating living functional heart tissue that can grow with the child. However, creating functional heart tissue in vitro remains a major challenge, as mature cardiomyocytes (CMs) are mostly non-proliferative (5). While embryonic and fetal CMs are highly proliferative and can restore function in damaged or diseased hearts (6, 7), the causes of decreased CM proliferation and loss of cardiac regenerative capacity after birth are still largely unknown. Elucidating factor that promote CM proliferation will greatly impact tissue engineering and regenerative approaches to treating CHD in children. The extracellular matrix (ECM) modulates a variety of cell functions, including proliferation (8), and there is evidence that ECM composition and stiffness of the heart change during development and maturation (9-12). The hypothesis of this project is that recapitulation of the fetal ECM environment will promote the proliferation of CMs; specifically, it is expected that the features of fetal cardiac ECM that promote CM proliferation are the cues that are lost or diminished in the adult heart. The hypothesis will be systematically tested in vitro, to determine the individual and combined effects of ECM composition and stiffness in 2D and 3D culture, and in vivo, as an initial step towards future clinical translation For ECM composition studies, native fetal and adult rat hearts will be decellularized to obtain ECM and then solubilized and adsorbed onto tissue culture dishes. Primary neonatal rat CMs will be seeded onto cardiac ECM-coated substrates and assayed for proliferation. In parallel, the effects of substrate stiffness will be determined. Decellularized and native hearts will undergo mechanical testing to determine the stiffness of the ECM. Polyacrylamide (PAAm) gels will be made with stiffnesses that mimic fetal and adult hearts. In these studies, Collagen I will be used at the same ligand density in fetal vs. adult stiffness gels to decouple composition from stiffness To investigate the combined effects of ECM composition and stiffness on CM proliferation, solubilized cardiac ECM will be incorporated into PAAm gels. The ECM/stiffness combination that results in highest CM proliferation will be used to guide the design of an injectable ECM-based biomaterial. The 3D gel will be characterized and optimized in vitro and will reveal whether 2D vs. 3D culture has different effects on CM proliferation. The ECM gel which results in greatest cell viability, infiltration, and proliferation in vitro will be tested in vivo for itseffects on stimulating CM proliferation in neonatal rats. The results of this work will significantly impact future cardiac tissue engineering approaches through the development and characterization of ECM-based biomaterials that promote CM proliferation and regeneration of cardiac tissue.